Pharmaceutical Composition Comprising a GLP-1-Agonist and Methionine

ABSTRACT

A liquid composition comprising a GLP-1 agonist or/and a pharmacologically tolerable salt thereof and, optionally, at least one pharmaceutically acceptable excipient, wherein the composition comprises methionine, as add-on therapy with metformin and/or with long-acting insulin/insulin derivates where appropriate.

The present application relates to a liquid composition comprising aGLP-1 agonist or/and a pharmacologically tolerable salt thereof and,optionally, at least one pharmaceutically acceptable excipient, whereinthe composition comprises methionine.

The present application further relates to the composition according tothe present invention for treating diabetes mellitus. The presentapplication further relates to the use of a composition according to thepresent invention in the manufacture of a pharmaceutical for treatingdiabetes mellitus. The present application further relates to a methodfor manufacturing a composition according to the present invention,comprising formulating a GLP-1 agonist or/and a pharmacologicallytolerable salt thereof with methionine and, optionally, at least onepharmaceutically acceptable excipient. The present application furtherrelates to a method for treating a patient with a composition accordingto the present invention, comprising administering the composition tothe patient.

Customary compositions of GLP-1 compounds comprise a tonicity modifier,a buffer for adjusting the pH, and a preservative.

WO2001/04156 (Zealand Pharmaceuticals) discloses a liquid composition ofSer³⁹-exendin-4(1-39)-NH₂), sodium dihydrogenphosphate, andpreservatives.

WO 2004/035623 (Zealand Pharmaceuticals) discloses a liquid compositioncomprising a stabilized exendin, 50 mM histidine, 100 to 200 mM sucrose,mannitol or other acceptable sugar, 20 mM methionine, 20 mMasparagine-glutamine or Asp, at a pH of 5.3. Stabilization is effectedby certain modifications of the amino acid building blocks ofexendin-4(1-39), for example, at positions Gln13, Met14, Trp25, orAsn28.

WO 2005/021022 (Novo Nordisk) discloses a liquid composition comprisingacetylated GLP-1, phenol as a preservative, mannitol and glycerol as atonicity modifier, and, optionally, a buffer.

WO 2006/051110 (Novo Nordisk) discloses liquid compositions comprisingliraglutide (GLP-1 compound), poloxamer 188 or poloxamer 407 (PluronicF-127) as a surface-active substance, phenol, propylene glycol, andsodium phosphate (pH 7.7). Addition of poloxamer-188 or poloxamer-407led to stabilization.

Exendins are a group of peptides which can lower blood glucoseconcentrations. Exendins have a certain similarity to the sequence ofGLP-1(7-36) (53%, Goke et al. J. Biol Chem 268, 19650-55). Exendin-3 andexendin-4 stimulate an increase in cellular cAMP production in theacinar cells of the guinea pig pancreas by interacting with exendinreceptors (Raufman, 1996, Reg. Peptides 61:1-18). Exendin-3, in contrastto exendin-4, effects an increase in the release of amylase in theacinar cells of the pancreas. Exendins act as GLP-1 agonists.

Glucagon-like peptide 1 (GLP-1) is an endocrine hormone which enhancesthe insulin response following oral intake of glucose or fat. Ingeneral, GLP-1 lowers glucagon concentrations, slows gastric emptying,stimulates (pro)insulin synthesis, enhances sensitivity to insulin, andstimulates insulin-independent glycogen synthesis (Hoist (1999), Curr.Med. Chem 6:1005, Nauck et al. (1997) Exp Clin Endocrinol Diabetes 105:187, Lopez-Delgado et al. (1998) Endocrinology 139:2811). Human GLP-1has 37 amino acid residues (Heinrich et al., Endocrinol. 115:2176(1984), Uttenthal et al., J Clin Endocrinol Metabol (1985) 61:472).Active fragments of GLP-1 include GLP-1 (7-36) and GLP-1(7-37).

Exendin-3, exendin-4 and exendin agonists have been proposed fortreating diabetes mellitus and preventing hyperglycemia, by reducinggastric motility and gastric emptying (U.S. Pat. No. 5,424,286 andWO98/05351).

Exendin analogs can be characterized by amino acid substitutions and/orC-terminal truncation of the native exendin-4 sequence. Such exendinanalogs are described in WO 99/07404, WO 99/25727, and WO 99/25728.

Solid-phase synthesis of AVE0010 is described in WO 01/04156 A1. AVE0010has the sequence: desPro³⁶exendin-4(1-39)-Lys₆-NH₂. This substance ispublished as SEQ ID NO:93 in WO 01/04156:

(SEQ ID NO: 1) H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-I-E-W-L-K-N-G-G-P-S-S-G-A-P-P-S-K-K-K-K-K-K-NH₂

Exendin-4 (39 AS) has the sequence:

(SEQ ID NO: 2) H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-I-E-W-L-K-N-G-G-P-S-S-G-A-P-P-P-S-NH₂

Exendin-3 has the sequence (J. Bio. Chem., 267, 1992, 7402-7405):

(SEQ ID NO: 3) H-His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro- Pro-Pro-Ser-NH₂

GLP-1 has the sequence:

(SEQ ID NO: 4) H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W-L-V-K-G-R-NH₂

It is an object of the present invention to increase the stability ofliquid formulations comprising a GLP-1 agonist. More particularly, it isan object of the present invention to improve physical and chemicalintegrity. We have found that this object is achieved by formulating theGLP-1 agonist with methionine.

It was found that methionine is able to increase the storage stabilityof a composition comprising a GLP-1 agonist such as AVE0010. Methioninedoes not affect the physical integrity of these compositions.

It was found that, surprisingly, the addition of methionine is able toimprove the storage stability of a composition according to the presentinvention by reducing the proportion of oxidation products ofmethionine, of proteins of high molecular weight, and of totalimpurities. These parameters are, individually or together, a measure ofthe chemical integrity of the compositions.

It was further found that, surprisingly, the biological activity of thecompositions according to the present invention is increased by theaddition of methionine.

The stability of pharmaceutically active polypeptides can be impaired byvarious mechanisms. These include pH, temperature, light, and theeffects of certain constituents.

A range of customary constituents of formulations of GLP-1 agonists canbe disadvantageous for the chemical or/and physical integrity and thestorage stability of formulations which comprise a GLP-1 agonist. Theseare, for example, polysorbate 20, polysorbate 80, poloxamer 188,benzalkonium chloride, and lysine. The compositions according to thepresent invention are therefore preferably free of these constituents.

The present invention accordingly provides for a liquid compositioncomprising a GLP-1 agonist or/and a pharmacologically tolerable saltthereof and, optionally, at least one pharmaceutically acceptableexcipient, wherein the composition comprises methionine.

The composition according to the present invention preferably comprisesmethionine in an amount ranging from 0.5 mg/mL to 20 mg/mL, morepreferably in an amount ranging from 1 mg/mL to 5 mg/mL. Methionine inthe D-form can be used. Likewise, methionine in the L-form can be used.Likewise, mixtures of the D-form and the L-form in any desiredproportions can be used.

More particularly, the composition according to the present invention isfree of surfactants, such as polyols and partial and fatty acid estersand ethers of polyhydric alcohols such as those of glycerol andsorbitol. The compositions according to the present invention are moreparticularly free of partial and fatty acid esters and ethers ofglycerol and sorbitol selected from the group consisting of Span®,Tween®, Myrj®, Brij®, Cremophor®. Furthermore, the compositionsaccording to the present invention are more particularly free of polyolsselected from the group consisting of polypropylene glycols,polyethylene glycols, poloxamers, Pluronics, Tetronics. Moreparticularly, the composition according to the present invention is freeof at least one substance selected from the group consisting ofpolysorbate, polysorbate and poloxamer.

More particularly, the composition according to the present invention issubstantially free, preferably free, of polysorbate, such as, forexample, polysorbate 20.

More particularly, the composition according to the present invention issubstantially free, preferably free, of polysorbate 80.

More particularly, the composition according to the present invention issubstantially free, preferably free, of poloxamer, such as, for example,poloxamer 188.

More particularly, the composition according to the present invention issubstantially free, preferably free, of benzalkonium chloride.

More particularly, the composition according to the present invention issubstantially free, preferably free, of histidine.

More particularly, the composition according to the present invention issubstantially free, preferably free, of EDTA, more particularly sodiumEDTA.

The composition according to the present invention can comprise one ormore substances which are customarily used to buffer the pH (buffersubstances).

Examples of such buffer substances are acetate, citrate, and phosphate,for example, in amounts of up to 5 mg/ml, up to 4 mg/ml, up to 3 mg/ml,or up to 2 mg/ml.

The composition according to the present invention can, likewise, besubstantially free of buffer substances. Likewise, the compositionaccording to the present invention can be free of buffer substances.

The composition according to the present invention can be substantiallyfree of citrate, acetate, and/or phosphate, or else free of citrate,acetate, and/or phosphate.

More particularly, the composition according to the present invention issubstantially free, preferably free, of histidine and sodium EDTA.

More particularly, no insulin is present in the composition according tothe present invention.

The pharmaceutical composition of the present invention can have anacidic or physiological pH. An acidic pH range is preferably in therange of pH 1-6.8, pH 3.5-6.8, or pH 3.5-5. A physiological pH ispreferably in the range of pH 2.5-8.5, more preferably pH 4.0 to 8.5,even more preferably pH 4.0 to 6.0. Especially preferred is a pH ofapproximately 4.5. For pH adjustment, physiologically safe dilute acids(typically HCl) and alkalis (typically NaOH) are suitable.

The composition according to the present invention can comprise asuitable preservative. Suitable preservatives are, for example, phenol,m-cresol, benzyl alcohol, and/or p-hydroxybenzoate esters, m-Cresol ispreferred.

Furthermore, the composition according to the present invention cancomprise suitable tonicity modifiers. Suitable tonicity modifiers are,for example, glycerol, dextrose, lactose, sorbitol, mannitol, glucose,NaCl, calcium or magnesium compounds such as CaCl₂ etc. Theconcentrations of glycerol, dextrose, lactose, sorbitol, mannitol, andglucose are customarily in the range of 100-250 mM, NaCl in aconcentration of up to 150 mM. Glycerol is preferred.

More particularly, the composition is intended for parenteraladministration. The composition according to the present invention ispreferably an injectable composition, more preferably for subcutaneousinjection. More particularly, the composition of the present inventionis suitable for injection once a day.

More particularly, the formulation according to the present inventionhas, after storage for 1 month, 2 months, 4 months, or 6 months at atemperature of +5° C. or 25° C., an activity of at least 80%, at least90%, at least 95%, or at least 98% of the activity at the start ofstorage.

In the present application, “activity” means the activity of the GLP-1agonist which is used in the formulation according to the presentinvention. Methods for determining the activity of a GLP-1 agonist areknown to a person skilled in the art.

Preferably, the composition according to the present invention has abiological activity of GLP-1 agonist of at least 89% or at least 90%after storage for 6 months at 25° C. The composition according to thepresent invention preferably has a biological activity of GLP-1 agonistof at least 45% or at least 50% after storage for 6 months at 40° C.

More particularly, the formulation according to the present inventionexhibits chemical integrity after storage for 1 month, 2 months, 3months, 4 months, or 6 months. Chemical integrity means, moreparticularly, that after storage at a temperature of +5° C., 25° C., or40° C. the formulation comprises at least 80%, at least 90%, at least95%, or at least 98% of the active substance, compared with the start ofstorage, in a substantially chemically unchanged form.

Chemical integrity can mean the chemical integrity of the GLP-1 agonist.GLP-1 agonists may comprise a methionine residue (e.g. position 14 inAVE0010). Chemical integrity of the GLP-1 agonist means, moreparticularly, that oxidation of the methionine residue is prevented.Here, chemical integrity means, more particularly, that the proportionof oxidized methionine with respect to the entire methionine content ofthe GLP-1 agonist after storage for 1, 2, 3, 4, or 6 months is below0.7%, below 0.6%, below 0.5%, below 0.4%, or below 0.3%. Storage can beeffected, for example, at 5° C., 25° C., or 40° C. Storage for 6 monthsat 5° C. is preferred, in which case the proportion of oxidizedmethionine is below 0.3%. Likewise, storage for 6 months at 25° C. ispreferred, in which case the proportion of oxidized methionine is below0.7%, below 0.6%, below 0.5%, below 0.4%, or below 0.3%. Likewise,storage for 6 months at 40° C. is preferred, in which case theproportion of oxidized methionine is below 1%, below 0.7%, below 0.6%,below 0.5%, below 0.4%, or below 0.3%.

Chemical integrity can mean a very low proportion of total impurities inthe formulation according to the present invention. The proportion oftotal impurities with respect to the entire mass of the GLP-1 agonistpresent in the formulation after storage for 6 months at 40° C. is moreparticularly below 50%, below 10% after storage at 25° C., or/and below1.8% after storage at 5° C.

Chemical integrity can mean a very low proportion of proteins of highmolecular weight in the formulation according to the present invention.The proportion of proteins of high molecular weight with respect to theentire mass of the GLP-1 agonist present in the formulation afterstorage for 6 months at 40° C. is more particularly below 5%, below 4%,below 3%, or below 2%. After storage for 6 months at 25° C., theproportion of proteins of high molecular weight with respect to theentire mass of the GLP-1 agonist present in the formulation is moreparticularly below 0.8%, below 0.7%, or below 0.6%.

More particularly, the formulation according to the present inventionexhibits physical integrity after storage for 1 month, 2 months, 4months, or 6 months. Physical integrity means, more particularly, thatafter storage at a temperature of +5° C., 25° C., or 40° C. theformulation comprises at least 80%, at least 90%, at least 95%, or atleast 98% of the active substance, compared with the start of storage,in a substantially physically unchanged form.

Physical integrity can mean the integrity of the GLP-1 agonist. Physicalintegrity means, more particularly, that the GLP-1 agonist does not formaggregates, such as, for example, fibrils.

The GLP-1 agonist is preferably selected from the group consisting ofexendin-3 and analogs and derivates thereof, exendin-4 and analogs andderivates thereof, and in which case the GLP-1 agonist is morepreferably selected from the group consisting of AVE0010 and exendin-4.

Exendin-3, analogs and derivates of exendin-3, exendin-4, and analogsand derivates of exendin-4 can be found in WO 01/04156, WO 98/30231,U.S. Pat. No. 5,424,286, EP application 99 610043.4, and WO 2004/005342.These documents are incorporated herein by reference. The exendin-3,exendin-4, and analogs and derivates thereof described in thesedocuments can be synthesized by means of the methods described therein,after which modifications are optionally carried out.

The sequences of AVE0010 (SEQ ID NO:1), exendin-4 (SEQ ID NO:2), andexendin-3 (SEQ ID NO:3) show a high degree of similarity. The sequencesof AVE0010 and exendin-4 are identical at positions 1-37. Sequence 1-39from exendin-4 is at 37 of the 39 positions (94%) identical to theexendin-3 sequence at positions 48-86. With reference to the sequences,a person skilled in the art can readily convert the positions specifiedherein, which relate to a particular sequence (e.g. to the sequence ofAVE0010 or exendin-4), to other sequences.

Analogs and derivates of exendin-3 or/and exendin-4 contain moreparticularly a modified amino acid sequence. For example, single aminoacids can be deleted (e.g. desPro36, desPro37, desAsp28, desMet(O)14 inexendin-4 and the corresponding positions in exendin-3). Likewise,single positions can be substituted (e.g. Met(O)¹⁴, Trp(O₂)²⁵, IsoAsp²⁸,Asp²⁸ Pro³⁸ in exendin-4 and the corresponding positions in exendin-3),in which case unnatural amino acids such as Met(O) (methionine sulfoxideor methionine sulfone), Trp(O₂) (N-formylkynurenine), or/and IsoAsp(β-aspartate or isoaspartate) can also be used. Unnatural amino acidscan be readily inserted, in the form of corresponding amino acidbuilding blocks, into the sequence.

Furthermore, the C-terminus or/and the N-terminus can be modified, forexample, by an additional sequence such as -(Lys)-, -(Lys)₂-, -(Lys)₃-,-(Lys)₄-, -(Lys)₅-, -(Lys)₆-, -Asn-(Glu)₅-, in which case -(Lys)₄-,-(Lys)₅-, -(Lys)₆-, -Asn-(Glu)₅- are preferred. The carboxyl group atthe C-terminus is preferably modified to an amide group (—NH₂).Optionally, modification of the C-terminus or/and of the N-terminus iscarried out as a further step after completion of synthesis.

Pharmaceutically tolerable salts can be manufactured in a further stepafter completion of the synthesis cycles of the method according to thepresent invention. The manufacture of pharmaceutically tolerable saltsof peptides is known to a person skilled in the art. A preferredpharmaceutically tolerable salt is acetate.

The GLP-1 agonist is preferably selected from the group consisting ofexendin-4, analogs and derivates of exendin-4, and pharmacologicallytolerable salts thereof.

A further preferred GLP-1 agonist is an analog of exendin-4 selectedfrom the group consisting of:

H-desPro³⁶-exendin-4-Lys₆-NH₂,

H-des(Pro^(36,37))-exendin-4-Lys₄-NH₂,

H-des(Pro^(36,37))-exendin-4-Lys₅-NH₂ and pharmacologically tolerablesalts thereof.

A further preferred GLP-1 agonist is an analog of exendin-4 selectedfrom the group consisting of:

desPro³⁶[Asp²⁸]exendin-4 (1-39),

desPro³⁶[IsoAsp²⁸]exendin-4 (1-39),

desPro³⁶[Met(O)¹⁴,Asp²⁸]exendin-4 (1-39),

desPro³⁶[Met(O)¹⁴,IsoAsp²⁸]exendin-4 (1-39),

desPro³⁶[Trp(O₂)²⁵,Asp²⁸]exendin-2 (1-39),

desPro³⁶[Trp(O₂)²⁵,IsoAsp²⁸]exendin-2 (1-39),

desPro³⁶[Met(O)¹⁴Trp (O₂)²⁵,Asp²⁸]exendin-4 (1-39),desPro³⁶[Met(O)¹⁴Trp(O₂)²⁵,IsoAsp²⁸]exendin-4(1-39) andpharmacologically tolerable salts thereof.

A further preferred GLP-1 agonist is an analog of exendin-4 selectedfrom a group as described in the previous paragraph, wherein the peptide-Lys₆-NH₂ is attached to the C-termini of the analogs of exendin-4.

A further preferred GLP-1 agonist is an analog of exendin-4 selectedfrom the group consisting of:

H-(Lys)₆-desPro[Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,

desAsp²⁸Pro³⁶,Pro³⁷,Pro³⁸exendin-4(1-39)-NH₂,

H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-NH₂,

H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-NH₂,

desPro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-desPro³⁶[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,

H-desAsp²⁸ Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵]exendin-4(1-39)-NH₂,

H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-NH₂,

H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-NH₂,

desPro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-Asn-(Glu)₅-desPro³⁶, Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-desPro³⁶[Met(O)¹⁴, Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,

desMet(O)¹⁴ Asp²⁸ Pro³⁶,Pro³⁷,Pro³⁸exendin-4(1-39)-NH₂,

H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-NH₂,

H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-NH₂,

desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,

H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-desPro³⁶[Met(O)¹⁴, Trp(O₂)²⁸,Asp²⁸]exendin-4(1-39)-Lys₆-NH₂,

desAsp²⁸Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴, Trp(O₂)²⁵]exendin-4(1-39)-NH₂,

H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁶,Asp²⁸]exendin-4(1-39)-NH₂,

H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]exendin-4(1-39)-NH₂,

desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂,

H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁶,Asp²⁸]exendin-4(1-39)-(Lys)₆-NH₂and pharmacologically tolerable salts thereof.

Likewise, the GLP-1 agonist can be selected from the group consisting ofGLP-1 and analogs and derivates of GLP-1. A further preferred GLP-1agonist is selected from the group consisting ofArg³⁴,Lys²⁶(N^(ε)(γ-glutamyl(N^(α)-hexadecanoyl)))GLP-1(7-37)[liraglutide] and a pharmacologically tolerable salt thereof.

A further preferred GLP-1 agonist is AVE0010. AVE0010 has the sequencedesPro³⁶exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:1). Likewise,pharmacologically tolerable salts of AVE0010 are preferred.

The GLP-1 agonist, for example AVE0010, is more particularly used in anamount ranging from 0.01 mg/ml to 0.5 mg/ml or 0.05 mg/ml to 1.5 mg/ml.

In a particular embodiment, the formulation according to the presentinvention comprises the following constituents:

-   (a) desPro³⁶exendin-4(1-39)-Lys₆-NH₂ (e.g. approximately 0.1 mg/mL),-   (b) sodium acetate trihydrate (approximately 3.5 mg/mL),-   (c) m-cresol (approximately 2.7 mg/mL),-   (d) L-methionine (approximately 3 mg/mL),-   (e) 85% glycerol (approximately 18 mg/mL),-   (f) approximately 0.1 N hydrochloric acid, if adjustment to a pH of    approximately 4.5 is required,-   (g) approximately 0.1 N NaOH solution, if adjustment to a pH of    approximately 4.5 is required, and-   (h) water.

More particularly, the formulation according to the present inventionconsists of the constituents mentioned in (a) to (h).

In the present application, “approximately” means that the constituentscan be present, for example, within the ranges of ±10, ±20, or ±30%around the specified values in the compositions according to the presentinvention.

If the composition according to the present invention comprises morethan one GLP-1 agonist, then these GLP-1 agonists are selectedindependently of one another.

Suitable packaging for the composition according to the presentinvention is, for example, a syringe or a glass vessel with a suitableclosure, from which individual therapeutically effective doses can bewithdrawn as needed. Equally suitable are injection pens foradministering doses; such pens comprise a container (e.g. a cartridge)which contains a pharmaceutical composition according to the presentinvention.

The present invention further provides for a method for treating apatient with a composition according to the present invention,comprising administering the composition to the patient.

The composition according to the present invention is intended moreparticularly for treating diabetes mellitus, more particularly fortreating type I or type II diabetes mellitus. Further possibleindications are symptoms which are associated with diabetes mellitus.Preferably, the composition according to the present invention is usedto control the fasting, postprandial, or/and postabsorptive plasmaglucose concentration, to improve glucose tolerance, to preventhypoglycemia, to prevent functional loss of the β-cells of the pancreas,to effect weight loss, or/and to prevent weight gain.

The present invention further provides for the use of a compositionaccording to the present invention in the manufacture of apharmaceutical for treating diabetes mellitus, more particularly type Ior type II, or/and the symptoms associated with it, as described herein.

The present invention further provides a method for manufacturing acomposition according to the present invention, comprising formulating aGLP-1 agonist or/and a pharmacologically tolerable salt thereof withmethionine and, optionally, at least one pharmaceutically acceptableexcipient.

The present invention further provides for the use of the compositionaccording to the present invention together with the administration ofmetformin, sulfonylurea, or glitazones, a long-acting insulin/insulinderivate, and/or a combination thereof, more particularly as an add-ontherapy in the administration of metformin.

The present invention further provides for the use of the compositionaccording to the present invention in patients whose blood sugar levelscannot be controlled sufficiently by the administration of metformin,sulfonylurea, or glitazones, a long-acting insulin/insulin derivate,and/or a combination thereof.

The present invention further provides for the use of the compositionaccording to the present invention in patients with type II diabetes asa supplement to a diet in order to improve blood sugar control.

More particularly, the composition comprises desPro³⁶exendin-4(1-39)-Lys₆-NH₂ (AVE0010), liraglutide and/or apharmacologically tolerable salt together with methionine and/or apharmacologically tolerable salt.

More particularly, Lantus®, N^(εB29)-tetradecanoyl des (B30) humaninsulin, or Insuman® is useful as a long-acting insulin derivative.

Especially preferred is the add-on therapy with metformin and/or along-acting insulin/insulin derivate and/or a pharmacologicallytolerable salt thereof for treating type II diabetes and/or obesity,more particularly in patients who are younger than 50 years and/or havea body mass index of at least 30.

In the present invention, the add-on therapy involves more particularlythe treatment of type II diabetes with metformin and AVE0010. Metforminand AVE0010 can be administered in a time interval of 24 hours.Metformin and AVE0010 can each be administered in a once-a-day dosage.Metformin and AVE0010 can be administered by means of different routesof administration. Metformin can be administered orally, AVE0010subcutaneously.

Patients treated with the add-on therapy according to the presentinvention can have an HbA1c value in the range of 7% to 10%. They arepreferably in the age range of 18 to 50 years.

The use in the add-on therapy according to the present invention is moreparticularly applicable to patients in whom type II diabetes cannot besufficiently controlled with metformin alone.

More particularly, metformin is administered as follows: at least 1.0g/day, preferably at least 1.5 g/day for 3 months.

The invention is further elucidated by the following examples andfigures.

LEGENDS

FIGS. 1 and 2 show the percentage content of oxidized methionine Met(ox)with respect to the entire methionine content of AVE0010 after storageat different temperatures. 1: start of storage t0. 2: storage for 1month. 3: storage for 3 months. 3: storage for 6 months. FIG. 1: batch894. FIG. 2: batch 897.

FIGS. 3 and 4 show the percentage content of protein impurities of highmolecular weight (with respect to AVE0010) after storage at differenttemperatures. 1: start of storage t0. 2: storage for 1 month. 3: storagefor 3 months. 3: storage for 6 months.

FIG. 3: batch 894. FIG. 4: batch 897.

FIGS. 5 and 6 show the percentage content of all impurities (withrespect to AVE0010) after storage at different temperatures. 1: start ofstorage t0. 2: storage for 1 month. 3: storage for 3 months. 3: storagefor 6 months. FIG. 5: batch 894.

FIG. 6: batch 897.

EXAMPLE 1

Liquid Composition Comprising AVE0010 and Methionine

The purpose of the study is the evaluation of the chemical or/andphysical stability of formulations of AVE0010 (solution for injection,0.1 mg/ml) with and without methionine, when the product is stored incartridges under long-term conditions and accelerated conditions for upto 6 months.

The following compositions are tested:

Composition A (2 parallel batches: AVE0010_09_894_A andAVE0010_09_897_A)

Specification according to Substance pharmacopeia Amount per unitAVE0010 Sanofi-Aventis 0.10 mg Sodium acetate trihydrate Ph. Eur./USP3.50 mg m-Cresol Ph. Eur./USP 2.70 mg 85% Glycerol Ph. Eur./USP 18.00 mg0.1N Hydrochloric acid Ph. Eur./USP ad pH 4.5 0.1N NaOH solution Ph.Eur./USP ad pH 4.5 Water for injection (Wfl) Ph. Eur./USP ad 1.0 ml

Composition B (2 parallel batches: AVE0010_09_894_B andAVE0010_09_897_B)

Specification according to Substance pharmacopeia Amount per unitAVE0010 Sanofi-Aventis 0.10 mg Sodium acetate trihydrate Ph. Eur./USP3.50 mg m-Cresol Ph. Eur./USP 2.70 mg L-Methionine Ph. Eur./USP 3.00 mg85% Glycerol Ph. Eur./USP 18.00 mg 0.1N Hydrochloric acid Ph. Eur./USPad pH 4.5 0.1N NaOH solution Ph. Eur./USP ad pH 4.5 Water for injection(Wfl) Ph. Eur./USP ad 1.0 ml

The formulations are stored in units which are intended for clinicalstudies and for sales and distribution.

Term Description Cartridge for Cartridge, 3 ml colorless, type I glass(Ph. Eur.), injection pen SAP number 100922 Crimped lid and 7.5 mminserted within a Crimped lid: aluminum gray sealing disk Sealing disk(exterior): isoprene rubber, material number 7773/35 Sealing disk(interior): bromobutyl rubber, material number 4780/40 Type I closure(Ph. Eur./USP) SAP number 164571 Plunger 9.2 × 11 mm Bromobutyl rubber,black SAP number 120521

Storage times, storage conditions, time points are summarized in thefollowing table.

Test interval (months) Condition 0 1 3 6 Long-term storage +5 ± 3° C. xx x x Accelerated conditions (temperature, humidity) +25 ± 2° C./60 ± 5%RH x x x +40 ± 2° C./75 ± 5% RH x x x

The formulations are stored horizontally. RH means relative humidity.Time point 0 is the start of storage. The measurements at time point 0are used as a reference for all conditions tested. During the tests, thesamples are stored at +5±3° C.

The physical and chemical stability of the stored formulations isdetermined with the help of the following tests:

-   -   Description    -   Clarity of the solution and color thereof    -   pH    -   Chemical stability (purity and impurities, determined by HPLC,        more particularly the proportion of oxidation products and of        total impurities)    -   Proteins of high molecular weight, determined by HPSEC    -   Visible particles    -   Biological activity of the formulations

Results

The formulations were studied separately for the parallel batches (894and 897) with regard to the following parameters:

-   -   Biological activity of AVE0010. At 5° C. and 25° C., activity        after 6 months was at least 96% of initial activity. The        activities of the compositions according to the present        invention were greater than the activities of the comparative        compositions. At 40° C., activity after 6 months in the absence        of methionine was approximately 43%. In the presence of        methionine, activity was approximately 51% and thus clearly        greater than in the absence of methionine.    -   Oxidation products. Measurements were carried out on an HPLC        instrument (model: alliance) from Water Systems, using the 100%        peak area method. For separation, a gradient of 0.1% TFA and        acetonitrile as the mobile phase and a C18 reversed-phase column        (Jupiter) as the stationary phase were used. At 5° C., the        proportion of oxidized methionine Met(ox) in AVE0010 in the        absence of methionine was 0.3%. At 25° C., the proportion was in        the range of 0.6-0.8%, at 40° C. 1.3%. When the formulation        comprised methionine, the proportion of oxidized methionine was        distinctly lower. It was never more than 0.2% under all        conditions tested. At 25° C., the proportion was thus        approximately only ¼ to ⅓ of the content in the absence of        methionine, even at 40° C. approximately only ⅙ (see FIGS. 1 and        2).    -   Proteins of high molecular weight. At 5° C., the proportion was        between 0.1 and 0.3% and remained substantially unchanged during        the entire storage time. At 25° C., the proportion rose in the        absence of methionine to 0.9 and 1.3%, respectively. In the        presence of methionine, the proportion was 0.4 to 0.5% and thus        less than half as high. At 40° C., the proportion was in the        absence of methionine 5.4% and 6.2%, respectively, while it was        in the presence of methionine only 1.6 and 1.7%, respectively,        and thus clearly lower (see FIGS. 3 and 4).    -   Total impurities. At 5° C., total impurities rose over the        storage time of 6 months slightly from 1.2 to 1.8 or 1.9%        (absence of methionine). When methionine was present, the rise        was a little lower. At 25° C., a rise to 10.6% and 11.8%,        respectively, was observed. In the presence of methionine, the        values were below 10%. At 40° C., the proportion rose up to 54%        (without methionine). When methionine was present, the        proportion was approximately only 47% (see FIGS. 5 and 6).    -   The percentage values are the content values (percentage values        of impurities) of the oxidation products, of total impurities,        and of high-molecular-weight proteins (HMWP).    -   All values were determined by HPLC with the so-called 100%        method. Here, in particular, it involves reversed-phase HPLC (C        18 column), in which a gradient method was used for the mobile        phase:    -   a) 0.1% TFA, 15% ACN and b) 0.1% TFA, 75% ACN.    -   Detection at 215 nm (UV).    -   The high-molecular-weight proteins (HMWP) were detected by        HPSEC, described in European Pharmacopeia 6.0 for injectable        insulin preparations.

The data are summarized in the following tables:

Mean values: AVE0010_09_894_A + B AVE0010_09_894_A AVE0010_09_894_B t₀ 1Mon. 3 Mon. 6 Mon. t₀ 1 Mon. 3 Mon. 6 Mon. 5° C. Total impurities 1.21.5 2.1 1.8 1.1 1.3 1.5 1.7 AVE0010 Test 101.5 99.6 98.0 97.8 101.1100.5 99.4 98.6 Proteins of high molecular weight 0.3 0.3 0.4 0.3 0.20.2 0.2 0.3 Oxidation products 0.3 0.4 0.4 0.3 0.1 0.2 0.1 0.1 25° C.Total impurities 1.2 3.0 6.4 11.8 1.1 2.5 5.7 9.8 AVE0010 Test 101.597.9 94.0 88.6 101.1 98.7 94.8 90.9 Proteins of high molecular weight0.3 0.4 0.6 1.3 0.2 0.3 0.3 0.5 Oxidation products 0.3 0.4 0.5 0.8 0.10.2 0.2 0.2 40° C. Total impurities 1.2 13.4 34.3 54.1 1.1 12.1 30.446.8 AVE0010 Test 101.5 87.1 66.6 42.5 101.1 88.8 70.8 50.9 Proteins ofhigh molecular weight 0.3 1.0 2.6 6.2 0.2 0.5 0.9 1.7 Oxidation products0.3 0.6 0.9 1.3 0.1 0.2 0.2 0.2

Mean values: AVE0010_09_897_A + B AVE0010_09_897_A AVE0010_09_897_B t₀ 1Mon. 3 Mon. 6 Mon. t₀ 1 Mon. 3 Mon. 6 Mon. 5° C. Total impurities 1.21.6 1.8 1.9 1.0 1.3 1.5 1.7 AVE0010 Test 99.2 98.2 97.5 96.7 99.5 99.298.0 97.1 Proteins of high molecular weight 0.1 0.1 0.1 0.2 0.1 0.1 0.10.1 Oxidation products 0.3 0.3 0.3 0.3 0.1 0.1 0.1 0.1 25° C. Totalimpurities 1.2 3.3 6.7 10.6 1.0 2.7 5.8 9.1 AVE0010 Test 99.2 96.6 92.887.4 99.5 97.8 93.6 90.0 Proteins of high molecular weight 0.1 0.2 0.50.9 0.1 0.1 0.2 0.4 Oxidation products 0.3 0.4 0.5 0.6 0.1 0.1 0.2 0.240° C. Total impurities 1.2 13.1 33.5 53.9 1.0 11.8 29.8 47.0 AVE0010Test 99.2 86.8 66.5 42.6 99.5 88.0 70.7 51.0 Proteins of high molecularweight 0.1 0.8 2.2 5.4 0.1 0.4 0.9 1.6 Oxidation products 0.3 0.5 0.81.3 0.1 0.1 0.2 0.2

CONCLUSION

The proportion of oxidation products, of proteins of high molecularweight, and of total impurities are, individually or together, a measureof the chemical integrity of the compositions. From the resultsdescribed above with the example compositions, it follows that theliquid compositions according to the present invention comprising

-   -   a GLP-1 agonist or/and a pharmacologically tolerable salt        thereof (more particularly AVE0010 or/and a pharmacologically        tolerable salt thereof),    -   optionally at least one pharmaceutically acceptable excipient,    -   and methionine

have improved stability or/and chemical integrity. The proportion ofoxidized methionine, of total impurities, and of proteins of highmolecular weight is lower in the compositions according to the presentinvention than in the comparative compositions. The compositionaccording to the present invention (batches 894_B and 897_B) and thecomparative compositions (batches 894_A and 897_A) differ in thepresence/absence of methionine. Therefore, improved stability or/andchemical integrity can be ascribed to the methionine constituent in thecompositions according to the present invention.

EXAMPLE 2

In a further experiment, it was studied how sodium EDTA and histidinehave an effect in a composition according to the present invention.

Composition B (as in Example 1)

Specification according to Substance pharmacopeia Amount per unitAVE0010 Sanofi-Aventis 0.10 mg Sodium acetate trihydrate Ph. Eur./USP3.50 mg m-Cresol Ph. Eur./USP 2.70 mg L-Methionine Ph. Eur./USP 3.00 mg85% Glycerol Ph. Eur./USP 18.00 mg 0.1N Hydrochloric acid Ph. Eur./USPad pH 4.5 0.1N NaOH solution Ph. Eur./USP ad pH 4.5 Water for injection(Wfl) Ph. Eur./USP ad 1.0 ml

Composition C

Specification according to Substance pharmacopeia Amount per unitAVE0010 Sanofi-Aventis 0.10 mg Sodium acetate trihydrate Ph. Eur./USP3.50 mg Sodium EDTA Ph. Eur./USP 1.00 mg m-Cresol Ph. Eur./USP 2.70 mgL-Methionine Ph. Eur./USP 3.00 mg L-Histidine Ph. Eur./USP 3.10 mg 85%Glycerol Ph. Eur./USP 18.00 mg 0.1N Hydrochloric acid Ph. Eur./USP ad pH4.5 0.1N NaOH solution Ph. Eur./USP ad pH 4.5 Water for injection (Wfl)Ph. Eur./USP ad 1.0 ml

In a standard experimental design, rabbits were treated with compositionB or C or a saline solution subcutaneously (s.c.) or intramuscularly(i.m.). In each case, half the rabbits were sacrificed after 24 hours or120 hours in order to determine the acute or subacute effects of theadministration histologically. Also, it was determined whetherrepair/regeneration of any changes occurred.

Following subcutaneous injection of composition C, the animals showedafter 24 hours, in contrast to the saline control, a light to moderateinflammatory reaction in the subcutaneous connective tissue. Aftersubcutaneous injection 120 hours earlier, a clear trend was observablefor the observed changes to repair by a fibroblastic reaction. Thus,compatibility could still be rated as moderate (instead of asincompatible).

With composition B, the animals showed after subcutaneous injection noor minimal differences to the saline control (good compatibility).

After intramuscular injection of composition C, the animals exhibitedmuscular necrosis (multifocal or disseminated), clearly differing fromthe saline controls, in which only the site of injection was visible asa clearly circumscribed necrotic area. With composition C,mineralization of the necrotic muscular tissue was observed after 120hours, visible even in a necropsy of the animals. Although small orfocal mineralization at various sites in rabbits is not unusual, themineralization after injection of composition C was clearly associatedwith the necrotic areas. Thus, the reversibility of the lesions causedby the injection is more than questionable. Based on these findings,composition C after intramuscular injection in rabbits was rated asincompatible.

Composition B after intramuscular injection showed good compatibility(no or minimal differences to the saline control).

From these data, it follows that composition B, compared to compositionC, had an improved compatibility in intramuscular or subcutaneousadministration. Subcutaneous injection is the preferred route ofadministration for the compositions comprising a GLP-1 agonist, moreparticularly AVE0010, described in this application.

Thus, the compositions according to the present invention, whichcomprise a GLP-1 agonist, more particularly AVE0010, can be free of EDTAor/and histidine. Likewise, the compositions according to the presentinvention can be substantially free of EDTA and histidine.

1. An aqueous liquid composition consisting essentially of: (a)desPro³⁶exendin-4(1-39)-Lys₆-NH2 (“AVE0010”) or a pharmacologicallytolerable salt thereof at a concentration of about 0.025 mg/mL to about0.1 mg/mL; (b) insulin glargine or a pharmacologically tolerable saltthereof at a concentration of about 3.64 mg/mL; (c) L-methionine; and(d) water.
 2. The aqueous liquid composition of claim 1, wherein thecomposition exhibits chemical integrity after storage for 6 months at atemperature of +25° C.
 3. The aqueous liquid composition of claim 2,wherein at least 80% of the liquid composition's active substance is ina substantially chemically unchanged form after storage for 6 months ata temperature of +25° C.
 4. The aqueous liquid composition of claim 2,wherein the percentage of oxidized methionine with respect to the entiremethionine content of the GLP-1 agonist after storage for 6 months at atemperature of +25° C. is below 0.7%.
 5. The aqueous liquid compositionof claim 2, wherein the proportion of total impurities with respect tothe entire mass of the GLP-1 agonist present in the composition afterstorage for 6 months at +25° C. is below 10%.
 6. The aqueous liquidcomposition of claim 2, wherein the proportion of proteins of highmolecular weight with respect to the entire mass of the GLP-1 agonist inthe composition after storage for 6 months at +25° C. is below 0.8%. 7.An aqueous liquid pharmaceutical composition comprising the followingconstituents per 1 mL volume of the composition: (a) approximately 50 μgdesPro³⁶exendin-4(1-39)-Lys₆-NH₂ (“AVE0010”) or a pharmacologicallytolerable salt thereof; (b) approximately 3.5 mg sodium acetatetrihydrate; (c) approximately 2.7 mg m-cresol; (d) approximately 3.0 mgL-methionine; (e) approximately 18.0 mg glycerol; and (f) water, whereinthe composition has improved stability and chemical integrity; andwherein the composition has improved compatibility in intramuscular orsubcutaneous administration.
 8. An aqueous liquid pharmaceuticalcomposition comprising the following constituents per 1 mL volume of thecomposition: (a) approximately 100 μg desPro³⁶exendin-4(1-39)-Lys₆-NH₂(“AVE0010”) or a pharmacologically tolerable salt thereof; (b)approximately 3.5 mg sodium acetate trihydrate; (c) approximately 2.7 mgm-cresol; (d) approximately 3.0 mg L-methionine; (e) approximately 18.0mg glycerol; and (f) water, wherein the composition has improvedstability and chemical integrity; and wherein the composition hasimproved compatibility in intramuscular or subcutaneous administration.